Inhibition of proteases is an increasingly important approach in the control of pathogenic organisms, including retroviruses. Such approaches may also be important in the preparation, processing, and maintenance of various biological materials; vaccine stability and cold-chain independent transport may be facilitated by such methods.
Retroviruses produce a polycistronic mRNA that encodes precursor molecules for the structural and functional viral proteins. A virally encoded aspartic acid protease is responsible for the processing of the polyprotein precursors Gag and Gag-Pol into the mature structural and replication enzymes. A gag-pol polyprotein homodimer forms to generate the Protease catalytic active site. Protease is then released from the precursor in an autocatalytic process. This processing is a critical step in the life cycle of retroviruses, including human immunodeficiency virus ("HIV"), the etiological agent of acquired immunodeficiency syndrome ("AIDS"). Improper processing, premature activation of the protease, or partial inhibition of the enzymatic activity during viral replication leads to defects in viral assembly and the formation of non-infectious, aberrant virus particles.
The HIV protease is autocatalytic, releasing itself from the precursor molecule by cleavage at two sites in the precursor polyprotein open reading frame ("ORF"); the amino ("N-") terminal extension of the Protease is removed, followed by cleavage at the carboxyl ("C-") terminus (Strickler et al. (1989), Proteins 6:139-154). Analysis of viral mutants suggested that the N- and C- cleavages are interdependent (Louis, J. M. (1991), Euro. J. Biochem. 199:361-369; Louis, J. M., et al. (1991), Adv. Exp. Med. Biol. 306:499-502). In addition, autoprocessing at the C- cleavage site is not significantly affected by the presence of the N-terminal precursor sequence. (Valverde et al. (1992), J. Gen. Virology 73:639-651).
A two step mechanism for autoprocessing of the HIV protease precursor polyprotein has been proposed (Louis, J. M., et al. (1994), Proc. Natl. Acad. Sci. USA, 91:7970-7974), wherein a first step is N-terminal Protease cleavage, releasing an active protease-polymerase intermediate(s), followed by a second step of release of mature Protease enzyme by cleavage at the C-terminus. The HIV protease autoprocessing suggests that HIV regulates protease expression in order to prevent premature complete cleavage (Arrigo, S. J., et al. (1995), DNA and Cell Biology, 14:15-23). It has also been noted that deletion of one region of the polyprotein precursor, the p6 region, enhances processing. (Partin, K. et al. (1991), Proc. Natl. Acad. Sci. USA 88(11):4776-4780).
Proper processing at HIV Gag and Gag-Pol cleavage sites is crucial for viral infectivity (Robins and Plattner, (1993) J. AIDS 6:162-170). Cleavage sites in Gag-Pol include inter alia:
p17/p24/p2/p7/p1/p6/p51/p15/p34. (See Table I, and see FIG. 1; see also Table I, Methods in Enzymology, 241:265, (Eds. L. C. Kuo and J. A. Shafer); Dunn, B. M., et al. (1978), J. Biol. Chem. 253:7269-7276).
The Gag and Gag-Pol cleavage sites are a prime target for protease inhibitors. For example, peptide-based HIV protease maturation inhibitors have been generated that target these sites in order to interfere with HIV processing (Burgess, K. and Pal, B. (1994), Bioorganic & Med. Chem. 2:23-26). Gag and Gag-Pol cleavage site sequences have also been modified to generate peptide-analog inhibitors (Marshall, G. R. et al., U.S. Pat. No. 5,342,922; Burgess, K. and Pal, B. (1994), Bioorganic & Med. Chem. 2:23-26). In addition, the p7/p6 junction of Gag has been identified as a potential target for protease inhibitors (Billich, S. et al. (1988), J. Biol. Chem. 263:17905-17908; Roberts, N. A., et al. (1990), Science 248:358-361). However, to date, very little attention has been given to the structural or functional significance of the transframe ("TF") region of the viral polyprotein.
Substrate competition is another approach for protease inhibition; target peptides corresponding to protease active site substrates compete for access to the mature enzyme recognition site. In addition, peptides and peptide analogs which mimic substrate intermediates of enzyme-catalyzed hydrolytic reactions have been described as aspartyl protease inhibitors (Rich, D. L. (1986) Proteinase Inhibitors, (Barrett and Salvesson, Eds.) Elsevier Science Publishers BV). Furthermore, such peptide inhibitors have been altered preventing hydrolysis of the substrate peptide bonds, thereby blocking the protease from releasing the substrate peptide and hydrolyzing the true target sequence. For example, such peptides have been used to inhibit HIV virus activity and to inhibit the proliferation of HIV in infected human lymphocytes (Voges, K. P., et al., U.S. Pat. No. 5,145,951). A limitation to this approach is that the inhibitor peptide may still be released from its complex with the protease and again become available for processing of the true target sequence.
Poor water solubility of protease inhibitors further impairs the in vivo utility of HIV peptide-based protease inhibitors (Robins, T. and Plattner, J. (1993), J. AIDS 6:162-170), and has been addressed by the addition of solubilizing groups such as poly-lysine, arginine methyl ester, glutamic acids, aspartic acids, Asp-Arg, Gly-Lys-Lys and dextran in order to improve the solubility of some inhibitory compounds (Toniolo, C., et al. (1994), J. Med. Chem. 37:4558-4562; Hostetler, K. Y., et al. (1994), Biochem Pharmacol 48:1399-1404). The present invention overcomes such limitations without requiring chemical modification strategies.
Thus, present day approaches cannot completely block virus activity or the resulting virus-induced pathology. Previous reports fail to identify a useful mechanism for inhibition and control of HIV protease maturation as disclosed in the present invention.
Furthermore, up to the present, no satisfactory treatment has been available which is based solely on inhibition of the mature viral protease. Current approaches to protease inhibition rely on peptides that are hydrophobic and which are directed to specific sequences of the active site in order to competitively inhibit protease activity. Such approaches are reported to result in the rapid selection of viral variants that are resistant to such inhibitors (Winslow, D. L. and Otto, M. J. (1995), AIDS 9 (suppl A):S183-S192).
It is therefore an object of the present invention to develop a method of inhibition of a virus encoded protease by inhibiting the maturation of the enzyme. Another object of the present invention relates to isolated forms of naturally occurring, virally encoded inhibitory peptide sequences which regulate protease maturation, arrest activation and/or reduce catalytic activity of the protease.
It is a further object of the present invention to combine competitive inhibition of protease activity with inhibition of protease maturation and therefore inhibition of activation of the protease itself. This approach will avert the rapid selection of viral variants that are resistant to the inhibitor.
Another object of the present invention relates to use the inhibitory peptides in a screening assay system of test compounds wherein additional, potent as partial protease inhibitors may be identified that also block protease activity either by competitive inhibition or by inhibition of protease maturation.
Because the natural inhibitory region is important in the control and regulation of the virus life cycle, it is a further object of the present invention to use isolated peptides derived from the inhibitory region in a formulation to stabilize a virus preparation, for example, as part of a stablilized vaccine preparation, by a method of inhibition of protease induced degradation or processing of retroviral virions. The instant TF peptide inhibitors are further packaged in the virus particle to maintain the protease and block autolysis of the protease for function early during virus infection.
Yet another object of the present invention is to use antibodies and anti-idiotypic antibodies against the inhibitor element to inhibit virion maturation and for direct treatment of virus-infected cells.